The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides and is currently being used in many different vaccine formulations called conjugate vaccines. Diphtheria toxin is a protein exotoxin produced by the bacterium Corynebacterium diphtheriae upon infection with the phage 0197. Both, Diphtheria toxin (“DT”) and CRM197 are components of many vaccines, like for example against Bordatella pertussis, Clostridium tetani, C. diphtheriae, hepatitis B virus, and Haemophilus influenza type B (WO 9324148, WO 9700697, WO 02055105). In addition there has been a growing interest in CRM197 because of its potential antitumor activity relating to its capacity to bind the soluble form of HB-EGF (US 2006/0270600A1).
CRM197 is produced by C. diphtheriae infected by the non-toxigenic phage β197tox. β197tox was created by nitrosoguanidine mutagenesis of the toxigenic corynephage β (Uchida, T. et al. 1971, Nature New Biology 233:8-11). The CRM197 protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. DT has a mass of 58.350 kDa (CRM197=58.415 kDa) and consists of the N-terminal A and the C-terminal B domains (21 and 37 kDa) which are linked by a disulfide bridge connecting Cys186 and Cys201. The A fragment is toxic after being released from its disulfide-bonded partner, the B fragment. Nicking of the holotoxin by mild proteolysis at the connecting peptide at positions 191-3 is a prerequisite for the A fragment activation. The B fragment has no apparent enzymatic activity but is required for toxicity, probably due to targeting the holotoxin to the target cell membranes (Broker M, Costantino P, De Tora L, McIntosh E D, Rappuoli R: Biochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: use as a conjugation protein in vaccines and other potential clinical applications. Biologicals, 2011, 39(4):195-204.)
Infected C. diphtheriae cultures secrete the CRM197 protein across the cytoplasmic membrane out of the cell into the culture medium. The CRM197 protein has about the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid for glycine, G52E) in the mature protein and eliminates the toxic properties of diphtheria toxin (Giannini G, Rappuoli R, Ratti G: The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res 1984, 12(10):4063-4069).
Methods of preparing DT and CRM197 are described in U.S. Pat. No. 4,709,017, U.S. Pat. No. 5,843,711, U.S. Pat. No. 5,601,827, and U.S. Pat. No. 5,917,017. There are currently three different systems used for industrial preparation of CRM197. Two systems are based on the use of phage infected C. diphtheriae cells. The most recent development constitutes a recombinant expression system in Pseudomonas fluorescens. The method employs a secretion approach to the periplasm in a genetically optimized P. fluorescens strain using a CRM197 gene equipped with a signal peptide for secretion into the periplasm (US20110287443).
For example, diphtheria toxin is isolated from cultures of C. diphtheriae strain C7 (B197) and/or C. diphtheriae strain C7 (B197) pPx350 grown in a casamino acids and yeast extract-based medium under aerobic conditions. Adjustment of media components were shown to improve yields (U.S. Pat. No. 4,925,792, WO 2006 100108). CRM197 or DT are harvested from the supernatant of the culture, and concentrated by ultrafiltration. Ammonium sulfate precipitation is a first, and anionic exchange chromatography a second purification step.
However, production of significant quantities of the CRM197 protein for use in vaccines has been hindered due to low protein abundance (WO 2006 100108).
Techniques have been developed to bolster the production of CRM proteins using double lysogens (Isolation and characterization of C. diphtheriae nontandem double lysogens hyperproducing CRM197. R Rappuoli, Appl. Environ, Microbiol. September 1983 46:560-564; U.S. Pat. No. 4,925,792 issued to R. Rappuoli; and Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox− into two attachment sites on the C. diphtheriae chromosome. R Rappuoli, J L Michel, and JR Murphy; J. Bacteriol. March 1983 153:1202-4210) of the nontoxigenic corynephage β197. Rappuoli reports yields of CRM197 from double and triple lysogens up to three fold higher than from the single lysogens. The production levels of CRM197 by single lysogens are adequate but economically unsatisfactory for the production of vaccines which utilize CRM197 protein. It is important to note that the construction of double and triple lysogenic strains in order to increase expression efficiency in C. diphtheria e is a long process which requires a laborious screening phase.
Plasmids were developed for recombinant expression of CRM197 in C. diphtheriae (U.S. Pat. No. 5,614,382, 1995/5614382_1997). This makes it possible to increase the number of copies of the gene (up to 5-10 per cell) without having to select pluri-lysogenic bacterial strains.
As in the case of the Corynebacterium strains infected by the phage β197tox, CRM197 is expressed in special culture media with a low ferrous content. Despite a reduction in the amount of time required for the genetic handling of the bacterial strain, the output of CRM197 does not increase dramatically by comparison with the use of double lysogenes.
Alternative expression host cells for DT included a Salmonella typhi vaccine strain cvd 908-htra (Orr N, Galen J E, Levine M M: Expression and immunogenicity of a mutant diphtheria toxin molecule, CRM197, and its fragments in S. typhi vaccine strain CVD 908-htrA. Infect Immun 1999, 67(8):4290-4294). Salmonella is a Gram negative bacterium and similar expression host as E. coli. Expression levels from various constructs (with, without signal peptide) in cvd 908-htra were low and solubility and immunogenicity were poor. Utilizing the alternative, non-Sec dependent translocation system of the hemolysin operon improved expression of soluble DT, but levels were still low.
Reports for production of CRM197 in E. coli show low yields of soluble CRM197 and formation of insoluble product in inclusion bodies. Truncation approaches have been used in an attempt to enhance expression to higher levels. (Bishai W R, Miyanohara A, Murphy J R: Cloning and expression in E. coli of three fragments of diphtheria toxin truncated within fragment B. Journal of Bacteriology 1987, 169(4):1554-1563)
A single strand expression plasmid for CRM197 containing the mutated diphtheria toxin gene encoding CRM197 was used for expression in E. coli (Bishai W R, Rappuoli R, Murphy J R: High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. Journal of Bacteriology 1987, 169(11):5140-5151; Bishai 1987). In this publication, transcription of CRM197 was controlled by the endogenous and constitutive Ptox promotor. In addition, DT C-terminally fused to the alpha melanocyte stimulating hormone (“ABM508”) was expressed by the heat inducible PLambda promoter or the Ptac promoter for expression.
Bishai 1987 speculated that jamming of the secretion apparatus due to high level protein induction caused a growth stop after induction of expression of periplasmic DT/CRM197 variants. This can be a general problem in periplasmic protein expression that has been observed and resulted in low volumetric yields of protein previously (Benson S A, Hall M N, Silhavy T J: Genetic analysis of protein export in E. coli K12. Annual Review of Biochemistry 1985, 54:101-134). Jamming of the secretion apparatus and formation of insoluble protein suggested an inability of the E. coli cells to provide a productive translocation and folding environment for CRM197 biogenesis.
As a consequence, Bishai 1987 reasoned that cytoplasmic expression would avoid the translocon jamming. Thus, Bishai 1987 removed the signal peptide for directing the expression into the cytoplasm. Only at low temperatures and when cytoplasmic proteases were deleted, did the cytoplasmic expression constructs yield soluble product. Production was inefficient and lead to aggregates at elevated temperatures, and when proteases were present.
Bishai 1987 failed to show production of high levels of soluble protein CRM197 fusion protein, i.e., with signal peptide for periplasmic targeting. In a Coomassie stained SDS polyacrylamide gel, extracts containing the ABM508 expression construct showed an intense protein band corresponding to ABM508, whereas cells expressing CRM197 expressed from the natural promoter with the wild type signal peptide do not show an obvious band for CRM197 at the expected size of 58 kDa.
Thus, periplasmic expression was not considered an efficient production strategy and to date there is no efficient E. coli periplasmic expression system established for production of soluble and correctly folded CRM197 or DT.
A production system which is based on the cytoplasmic expression of insoluble CRM197 in inclusion bodies followed by solubilization, purification and refolding of the protein is provided by the first E. coli based expression system for CRM197 (WO2010 150230). Wild-type CRM197 without additional amino acids can only be obtained with this system when an additional proteolysis step is applied.
Signal peptides induce protein secretion to the periplasm and have various effects on protein biogenesis. (Powers T, Walter P: Co-translational protein targeting catalyzed by the E. coli signal recognition particle and its receptor. The EMBO Journal 1997, 16(16):4880-4886.) (Schierle C F, Berkmen M, Huber D, Kumamoto C, Boyd D, Beckwith J: The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the E. coli periplasm via the signal recognition particle pathway. Journal of Bacteriology 2003, 185(19):5706-5713).